Ammonificins A and B, Hydroxyethylamine chroman
derivatives from a Cultured Marine Hydrothermal Vent
Bacterium, Thermovibrio ammonificans
Eric H. Andrianasolo,† Liti Haramaty,† Richard Rosario-Passapera,† Kelly Bidle,‡ Eileen
White,§,, Costantino Vetriani,† Paul Falkowski † and Richard Lutz †*
Center for Marine Biotechnology, Institute of Marine and Coastal Sciences, Rutgers, The
State University of New Jersey NJ 08901-8521
Department of Biology, Rider University, 2083 Lawrenceville Rd, Lawrenceville, New Jersey
08648
Center for Advanced Biotechnology and Medicine, Department of Molecular Biology and
Biochemistry, Rutgers, The State University of New Jersey, 679 Hoes Lane, Piscataway, New
Jersey 08854
University of Medicine and Dentistry of New Jersey, Robert Wood Johnson Medical School,
675 Hoes Lane, Piscataway, New Jersey 08854
The Cancer Institute of New Jersey, 195 Little Albany Street, New Brunswick, New Jersey
08903
*To whom correspondence should be addressed. Tel.: (732) 932-8959 ext. 242
Fax :(732) 932-6557 E-mail: rlutz@imcs.marine.rutgers.edu
†
IMCS, Rutgers, The State University of New Jersey.
‡
Department of Biology, Rider University
§
CABM, Rutgers, The State University of New Jersey.
 University

of Medicine and Dentistry of New Jersey.
The Cancer Institute of New Jersey.
1
Abstract
Two hydroxyethylamine chroman derivatives, ammonificins A (1) and B (2), were
isolated from the marine hydrothermal vent bacterium, Thermovibrio ammonificans.
The molecular structures of these compounds were determined using a combination of
NMR, mass spectrometry and CD analyses. Biological activities were determined using
an antimicrobial assay and the patented ApopScreen cell-based screen for apoptosisinduction and potential anticancer activity. Ammonificins A (1) and B (2) weakly induce
apoptosis in micromolar concentrations. Ammonificin B (2) is found to have
antimicrobial activity against Bacillus cereus. This is the first report of metabolites from
marine hydrothermal vent bacterium.
2
The oceans are the Earth’s largest ecosystem and hold great, underexplored
potential for drug discovery. Within this vast ecosystem, however, one area remains
enigmatic: deep-sea hydrothermal vents which are characterized by high concentration
of reduced sulfur compounds.1 Life is supported by the growth of chemolithoautotrophic
bacteria, capable of oxidizing hydrogen sulfide, hydrogen and other reduced inorganic
compounds to convert energy that is used to fuel carbon dioxide fixation into
macromolecules.
Chemoautotrophic organisms are known to produce chemical defenses against their
consumers.
Some
chemically
deterrent
species
are
also
known
to
harbor
chemoautotropic endosymbiotic bacteria, and these microbial symbionts may produce
metabolites that defend their host species.2 In this study we investigated the ability of
chemosynthetic bacteria to produce novel compounds capable of antimicrobial activities
and/or apoptosis induction.
As pathogens develop defenses against known antibiotics, they acquire an acute
resistance, which poses a serious problem for treating bacterial diseases. In recent
years, researchers have had diminishing success in identifying new antibiotics among
terrestrial organisms. Recently, a group of antibiotic molecules similar to streptomycin,
actinomycin and vancomycin was discovered from deep ocean sediment bacteria,
demonstrating that the oceans may present an alternative source for a new generation
of antibiotics.3-5 Chemotherapeutics currently in use for the treatment of cancer rely on
the ability to selectively target proliferating cells, which are enriched in tumors. Tumor
cells progressively evolve genetic mutations that enable not only cell proliferation, but
also resistance to apoptosis, a cell suicide pathway that is the cellular response to
3
oncogene activation or irreparable cellular damage
6-9.
Effective cancer therapeutic
strategies often rely on preferential and efficient induction of apoptosis in tumor cells.
Progressive exposure to such molecules commonly leads to selection of resistant cells
that are therapeutically associated with both tumor progression and resistance to
chemotherapy.6-9 While many conventional chemotherapeutics trigger apoptosis
indirectly by inflicting cellular damage, recent efforts have been directed toward
developing agents that specifically target or activate a caspase cascade that leads to
apoptosis.10
In our ongoing effort to discover and develop new drugs, we have screened extracts
from several marine organisms and have found that those produced from Thermovibrio
ammonificans, an anaerobic, chemolithoautotrophic marine bacterium isolated from a
deep-sea hydrothermal vent, possessed the ability to specifically induce apoptosis in
mammalian epithelial cells growth. Bioassay-guided purification resulted in the isolation
of two hydroxyethylamine chroman derivatives, ammonificin A (1) and B (2), which we
demonstrate are moderately capable of inducing apoptosis and inhibit Bacillus cereus
cell growth.
4
NH 2
OH
9
8
OH
8a
O
19
2
3
4a
5
O
4
HO
17
11
13
14
R
1 R= OH
2 R= Br
5
Br
Results and Discussion
Thermovibrio
ammonificans,
a
thermophilic,
anaerobic,
chemolithoautotrophic
bacterium, was isolated from the walls of an active deep-sea hydrothermal vent
chimney on the East Pacific Rise at 9° 50’ N. Cells of the organism were Gramnegative, motile rods that were about 1.0 µm in length and 0.6 µm in width. Growth
occurred between 60 and 80°C (optimum at 75°C), 0.5 and 4.5% (w/v) NaCl (optimum
at 2%) and pH 5 and 7 (optimum at 5.5). The generation time under optimal conditions
was 1.57 h. Growth occurred under chemolithoautotrophic conditions in the presence of
H2 and CO2, with nitrate or sulfur as the electron acceptor and with concomitant
formation of ammonium or hydrogen sulfide, respectively.11
Forty grams wet weight of the organism were extracted in methanol. One part of the
methanol soluble extract was dissolved in DMSO, and was tested for apoptosis
induction as assessed by the ApopScreen protocol.12,
13
This cell-based assay is
specific for the isolation and identification of apoptosis-inducing compounds. Two
genetically-matched immortal mouse epithelial cell lines are used: wild-type W2 with
apoptosis function intact, and D3 with apoptosis function disabled through specific
genetic deletion of both bax and bak (D3) (United States Patent #US 6,890,716). D3
cells are completely and irreversibly defective for apoptosis, and yet all apoptosis
regulatory mechanisms upstream of Bax and Bak (Bcl-2, Bim, etc.) remain intact.14
Importantly, the vast majority of human cancers with defects in apoptosis have the
pathway disabled upstream of Bax and Bak. Thus, screening to identify compounds
that have the capacity to kill W2 but not D3 cells should identify those that possess
proapoptotic,
and
potentially
anticancer,
6
activity.
Moreover,
materials
that
indiscriminately kill both apoptosis-competent W2 and apoptosis-defective D3 cells can
be used to eliminate those compounds that are non-specifically toxic.12
The extract that induced apoptosis (Fig. 4) was fractionated and subsequently
purified by analytical RPHPLC. Using this strategy, two pure compounds with
proapoptotic activity were isolated. Chemical structures of these two compounds (1 and
2) were ascertained by standard spectroscopic techniques, as described below.
The LR ESIMS of ammonificin A (1) displayed ion clusters at m/z 488(100)/490(98)
indicating the presence of one bromine. The molecular formula of 1 was established as
C23H22BrNO6 on the basis of HR ESIMS [m/z 488.0701 (M + H)+]. The 1H spectrum of 1
indicated clearly the presence of nine aromatic ring signals; H 6.67 [(d, J=7.9 Hz), H6], H 7.09 [(d, J=7.9 Hz), H-7], H 7.41 [s, H-12], H 6.77 [(d, J=7.2 Hz), H-15], H 7.26
[(d, J=7.2 Hz), H-16], H 6.65 [s, H-18], H 6.74 [(d, J=7.8 Hz), H-20], H 7.01 [(dd, J=7.8
Hz, 7.6 Hz), H-21], H 6.81 [(d, J=7.6 Hz), H-22]. Their corresponding methine carbons
were assigned from multiplicity edited HSQC: C-6 ( 108.0), C-7 ( 126.8), C-12 (
133.1), C-15 ( 118.4), C-16 ( 128.8), C-18 ( 101.4), C-20 ( 107.3), C-21 ( 130.5),
C-22 ( 106.8). Analysis of HMBC and multiplicity edited HSQC data suggested the
presence of nine quaternary carbons characteristic of signals belonging to aromatic ring
systems: (c 110.8, 116.1, 119.7, 137.8, 155.7, 156.9, 157.1, 157.9, and 158.9). Given
the number of carbon belonging to the aromatic signals, ammonificin A (1) is found to
possess three aromatic ring systems. Furthermore, three proton signals characteristic of
hydroxyl groups attached to aromatic ring systems were present in the 1H spectrum; H
8.48 (br s), H 9.26 (br s), H 9.47 (br s). Closer examination of the 1H-1H COSY along
with the 1H spectrum suggested the presence of signals characteristic of a dihydropyran
7
moiety: H 4.35 [(d, J=5.6 Hz), H-4], H 4.98 [m, H-3], H 4.45 [m, H-2]. HMBC
correlations between; H-6 and C-7 (c 126.8), C-5 (c 155.7), C-4a (c 116.1), H-7 and C8 (c 119.7), C-8a (c 157.9), H-4 and C-4a (c 116.1), H-2 and C-8a (c 157.9) strongly
suggested that 1 has a chroman moiety in its structure. Another interesting group
resulting from the 1H-1H COSY analysis is a hydroxyethylamine moiety15-17 in 1; H 4.70
[m, H-9], H 3.45 [m, H-10]. Moreover, this hydroxyethylamine moiety is found to be
attached to C-8 according to the HMBC correlation between H-9 and C-8 (c 119.7). The
two remaining aromatic rings were established using
1H-1H
COSY and HMBC
correlations (Figure 1). The 1H-1H COSY correlation between H-15 and H-16, HMBC
correlations between H-15 and C-11, C-13, C-14, C-16, and HMBC correlations
between H-12 and C-13, C-14, C-16 define a trisubstituted aromatic ring system. The
1H-1H
COSY correlations between H-21 and H-20, H-22, and HMBC correlations
between H-20 and C-19, C-22 and between H-22 and C-17, C-18, C-20 generated a
disubstituted aromatic ring system. The connectivity between the chroman moiety and
the remaining two ring systems was established by HMBC correlations: H-4 to C-12, C16 and H-3 to C-17. The chemical shift of the quaternary carbons belonging to the
aromatic ring systems played an important role in the assignment of the regiochemistry.
While both bromine and hydroxyl markedly deshield the carbon to which they are
attached, the effect of the hydroxyl is far larger. For example, the chemical shift of the
C-5 quaternary carbon (c 155.7) indicated that hydroxyl was attached whereas the shift
at the C-13 quaternary carbon (c 110.8) indicated bromine was present. Similarly, the
chemical shifts at C-14 (c 157.1) and C-19 (c 156.9) indicated that hydroxyls were
attached to these positions.
8
To determine the configurations at C-3, C-4 and C-9 a circular dichroism (CD)
spectrum of ammonificin A (1) was obtained. This experimental CD spectrum was then
compared to the predicted CD spectra of all possible stereoisomers. Theoretically
ammonificin A (1) has eight stereoisomers. The coupling constant between H-3 and H-4
(J=5.6 Hz) suggested a cis relationship between these two protons (H-3 equatorial, H-4
axial) which indicated of only four probable stereoisomers: (3S, 4S, 9R), (3S, 4S, 9S),
(3R, 4R, 9R), (3R, 4R, 9S).
These four probable stereoisomers were submitted to geometry optimization by DFT
(BLYP/6-31G*) approach18. For each minimized geometries a single CD spectrum was
calculated using TDDFT approach (B3LYP/TZVP).18 The overall CD spectra thus
obtained were subsequently UV-corrected and compared with the experimental one of
1. An excellent agreement between the particular CD curve calculated for 3S, 4S, 9R
and the experimental was found (Figure 2, top).This indicated that 1 has the following
configurations 3S, 4S, 9R, and the structure of 1 is established as shown.
The
LR
ESIMS
of
ammonificin
B
(2)
displayed
ion
clusters
at
m/z
550(51)/552(100)/554(48) indicating the presence of two bromines. The molecular
formula of 2 was established as C23H21Br2NO5 on the basis of HR ESIMS [m/z 549.9857
(M + H)+]. The molecular formula of 2 showed that it has one more bromine atom and
one less hydrogen and oxygen atom compared to 1.
The strong similarity of its 1H NMR spectrum to that of ammonificin A (1) revealed
that 1 and 2 share the same general structural features. Furthermore, only two proton
signals characteristic of hydroxyl groups attached to the aromatic ring system were
9
present in the 1H spectrum of 2 (H 9.27 (br s), H 9.46 (br s)), suggesting that one
hydroxyl group was replaced by one bromine atom. HMBC correlations between H-16
and C-14 and also between H-12 and C-14 confirm this suggestion. From the above
analyses, it is concluded that the structure of 2 is similar to that of 1 except that the
hydroxyl group attached to C-14 is replaced by one bromine atom. The configurations at
C-3, C-4 and C-9 of ammonificin B (2) were ascertained by the same methods as
described above.
NH 2
OH
OH
O
O
HO
Br
OH
COSY Correlations
HMBC Correlations
Figure 1. Key HMBC and selected COSY correlations for ammonificin A (1)
10
Figure 2. Comparison of the experimental CD spectrum (
calculated (
) for 3S, 4S, 9R (top) and 3R, 4R, 9S (bottom).
11
) of 1 with the spectra
Figure 3 Comparison of the experimental CD spectrum (
calculated (
) for 3S, 4R, 9S (top) and 3R, 4S, 9R (bottom).
12
) of 1 with the spectra
Figure 4. Comparison of the experimental CD spectrum (
calculated (
) for 3S, 4S, 9R (top) and 3R, 4R, 9S (bottom).
13
) of 2 with the spectra
In order to assess the pro-apoptotic activities of these compounds as a marker for
their potential anticancer efficacy, apoptosis induction potential was determined using
the ApopScreen protocol as described above. Apoptosis induction, in a dose response
manner (Figure 5), was detected in the presence of whole cell extract followed by
fractions and compounds. Due to the small amount of the ammonificins available for
ApopScreen, only three and one concentrations were tested for A and B, respectively.
At 252 μg/mL of whole cell extract, death of W2 cells was measured to be 52% and
growth of D3 of 57%. Addition of approximately 70.5 μM (46 μg/mL) of Fraction 2
resulted in 53% death of W2 and 77% growth of D3. In both experiments, upon addition
of
2.5 times or double (respectively) the apoptosis inducing concentration,
indiscriminate demise of both cell lines was measured, whereas an addition of half this
concentration had no effect on the viability of both cell lines. Fraction 4, at
approximately 514 μM (334 μg/mL), caused 15% death of W2, and 25% growth of D3.
Half this concentration had no effect on the cells.
ApopScreen results for ammonificin A (1) (purified from fraction 2) is also shown in
Figure 4. Incubation with 232 µM induced 21% death of W2, and D3 growth of 45%.
These results indicate that at this concentration the compound induces apoptosis as
defined for the ApopScreen assay. At 580 μM there was indiscriminate demise of both
cell lines, while no effect on viability was measured at 116 μM. Because of the small
amount of ammonificin B (2) (purified from fraction 4) available for ApopScreen, only
one concentration (182 µM) was tested, and was shown to induce apoptosis with death
of 17% for W2 and growth D3 of 41% (Figure 5). Untreated cells and staurosporine, a
known apoptosis inducer, were used as controls.
14
In the presence of 0.1 μM
staurosporine, W2 cells showed a death rate of 54%, and D3 growth of 65%. Thus, the
compounds we have isolated weakly activate apoptosis in immortalized mammalian
epithelial cells.
To determine if either ammonificin A (1) or B (2) also possessed any anti-microbial
properties, a dose-dependent growth analysis was performed using B. cereus and E.
coli. As seen in Figure 6, after two hours 50 μg of ammonificin B (2) reduced the
O.D.595 of B. cereus cultures by nearly 40% as compared with a control culture
containing no compound. Twice the amount of this same compound increased the
growth inhibition to reduce the O.D.595 of the culture by greater than two-thirds as
compared with the control. The effect of ammonificin A (1) on B. cereus growth was not
as severe; however, at a dose of 100 μg, there was a 30% reduction in cell density as
compared with the control.
For comparison, both ampicillin and kanamycin were
included as controls in these assays. Both of these antibiotics inhibited cell growth in B.
cereus to an extent comparable to the inhibition seen with ammonificin B (2), as
monitored by cell density (Fig. 6). Interestingly, the concentrations of ammonificin A (1)
and B (2) used in these analyses had no effect on cultures of E. coli (data not shown).
While these results are preliminary, they do offer compelling evidence that ammonificin
B (2) and, to a lesser extent, ammonificin A (1), have effective microbiocidal properties.
Future analyses will address examining both the anti-microbial effect of these
compounds on additional strains, including relevant human pathogens, as well as the
minimum inhibitory concentrations required for effectiveness.
15
The compounds described herein represent a potential platform for the development
of new drugs with specific proapoptotic activity, and therefore, potential anticancer utility
as well as a new anti-microbial class. This work describes the bioactivity of metabolites
from the marine hydrothermal vent bacterium Thermovibrio ammonificans, using a cellbased screen that we developed to identify potential proapoptotic compounds.
Thermovibrio ammonificans was used as a model organism; a better understanding of
its metabolism and genomics will provide a useful tool for future identification of
biosynthetic key pathways for secondary metabolism. The vast majority of human solid
tumors are of epithelial origin, and defects in apoptosis, mostly upstream of Bax and
Bak, play important roles in both tumor suppression and mediation of chemotherapeutic
responses. Consequently, efforts are increasingly focused on developing drugs that can
re-activate the apoptotic pathway. The compounds identified here induce apoptosis
upstream of Bax and Bak and may have a potential for use as anticancer agents that
exploit the apoptosis pathway in tumor cells.
16
Figure 5
Change in relative W2 (dark bars) and D3 (clear bars) cell viability by MTT assay,
48 h after addition of whole cell extract, Fraction 2 and 4, Ammonificin A and B, in a
range of concentrations. Staurosporine (0.1 μM), a known apoptosis inducer, and
untreated cells were used as positive and negative controls. Values were calculated
from an average of five wells.
17
1.2
1.0
0.8
0.6
0.4
0.2
0.0
0
50
100
150
200
µg/mL
Figure 6
Relative OD values, 595nm, of Bacillus cereus cultures 2 h after the addition of
Ammonificin A (dark squares) and B (clear squares), at various concentrations.
Ampicillin (x) and Kanamycin (+), known antibiotics, were used as controls. Each value
represents the average of three wells.
18
Experimental Section
General Experimental Procedures.
Optical rotations were measured on a JASCO P 1010 polarimeter. UV and FT-IR
spectra were obtained employing Hewlett-Packard 8452A and Nicolet 510 instruments,
respectively. CD spectra were acquired on JASCO J-720 spectropolarimeter. All NMR
spectra were recorded on a Bruker Avance DRX400 spectrometer, Varian-400
instrument (400 MHz) and Varian-500 instrument (500 MHz). Spectra were referenced
to residual solvent signal with resonances at δH/C 2.50 / 39.51 (DMSO-d6). ESI MS data
were acquired on a Waters Micromass LCT Classic mass spectrometer and Varian 500MS LC Ion Trap. HPLC separations were performed using Waters 510 HPLC pumps, a
Waters 717 plus autosampler, and Waters 996 photodiode array detector. All solvents
were purchased as HPLC grade.
Extraction and Isolation Procedures.
Cell culturing
Thermovibrio ammonificans
was routinely grown in modified SME medium as
previously described.11 For the purpose of this study, bacterial cells were harvested
from a total of 5 L of bacterial culture.
The material (40 g) was extracted three times with MeOH to give a polar crude
organic extract (550 mg). A portion of this extract (20 mg) was tested for apoptosis
induction. The crude organic extract was found active and subjected to fractionation
using solid phase extraction cartridge (normal phase silica) to give four fractions F1 to
F4 using hexane, hexane-EtOH, EtOH and MeOH as an increasingly hydrophilic solvent
19
system series. The fractions eluting with hexane-EtOH (F2) and MeOH (F4) had
apoptosis induction activity. These fractions were further chromatographed on analytical
RP HPLC (Phenomenex luna C18, 250 x 4.60 mm) using isocratic elution with 50%
MeOH and 50% H2O, flow rate 1 mL/min) to yield 3 mg of 1 (tR = 2.8 min) from F2, and
1.6 mg of 2 (tR = 4.4 min) from F4.
Computational Methods.
Geometry optimization, UV and CD computations were undertaken using TDDFT
with the B3LYP hybrid functional and a TZVP basis set, as included in the
TURBOMOLE suite of programs with TmoleX a graphical user interface to the
turbomole quantum chemistry program package.18 The corresponding oscillator and
rotatory strengths thus obtained were summed and energetically weighted, following the
Boltzmann statistics. Finally, the overall UV and CD spectra were simulated as sums of
Gaussian functions centered at the wavelengths of the respective electronic transitions
and multiplied by the corresponding oscillator or rotatory strengths, transformed into
absorption and Δε values, respectively.19-22
Biological Evaluation - Apoptosis Induction.
Apoptosis induction in the presence of compounds 1 and 2 was carried out as
described in Andrianasolo et al. 2007 using the ApoScreen assay. 13 In this assay
viability of treated W2 (apoptosis competent) and D3 (apoptosis defective)23 cells is
measured using a modification of the MTT assay24. For this study, viability was
measured at 0 and 48 h and differentail growth was calculated in the presence of the
compounds, Staurosporine, (an apoptosis inducer) as positive control, and DMSO as
negative controls.
20
Biological Evaluation – Anti-microbiol assay.
A dose-dependent growth analysis was performed on two strains of bacteria, Grampositive Bacillus cereus and Gram-negative Escherichia coli, to determine if
Ammonificin A (1) or B (2) possessed microbicidal properties. To initiate these tests,
overnight cultures of B. cereus and E. coli were diluted into fresh LB medium at an
O.D.595 of 0.1 in 96-well plates. A different concentration of each compound tested was
added to individual wells, in triplicate, and after 2 hours of incubation at 37°C bacterial
density was analyzed via spectroscopy. As a positive control to monitor inhibition of cell
growth, the antibiotics kanamycin and ampicillin were both included in these assays and
tested at a concentration of 50 µg/mL or 100 µg/mL, respectively.
Ammonificin A (1): UV (EtOH) max (log  ) 267 (2.90), 285 (3.68), 305 (3.70); CD
(EtOH) see Figure 2; IR
max
(neat) 3350, 2950, 1620, 1460, 1380, 1230, 1160, 1120,
1090, 1020, 805 cm-1; 1H NMR and 13C NMR, see table 2; HR ESIMS [m/z 488.0701 (M
+ H)+ (calcd for C23H23BrNO6, 488.0709)].
Ammonificin B (2): UV (EtOH) max (log  ) 267 (2.90), 285 (3.68), 305 (3.65); CD
(EtOH) see Figure 4; IR
max
(neat) 3350, 2950, 1620, 1460, 1380, 1230, 1160, 1120,
1090, 1020, 805 cm-1; 1H NMR and 13C NMR, see table 3; HR ESIMS [m/z 549.9857 (M
+ H)+ (calcd for C23H22Br2NO5, 549.9865)].
21
Acknowledgments
We thank K. McPhail and S. Kim for NMR data from NMR facilities at Oregon State
University and Department of Chemistry at Rutgers University, respectively. We also
thank H. Zheng for mass spectrometry analyses at the Center for Advanced
Biotechnology and Medicine, Rutgers University. This research was funded by Rutgers
University through an Academic Excellence award, by NSF grants OCE 03-27373
(R.A.L and C.V.) and MCB 04-56676 (C.V.), by the New Jersey Agricultural and
Experiment Station (C.V.), and by NIH grant R37 CA53370 (E.W.).
Supporting Information
NMR, MS data of 1 and 2. This material is available free of charge via the Internet at
http://pubs.acs.org.
22
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(24) Denyer, S. P.; Maillard, J.-Y. J. Immunol. Meth. 1983, 65, 55-63.
24
Table 2. NMR Spectroscopic Data of Ammonificin A (1) (400 MHz, DMSOd6)
Position
C
H
HMBCa
2
70.2, CH2
4.45, m
8a, 4
3
83.3, CH
4.98, m
17
4
27.5, CH
4.35, d (5.6)
4a, 12, 16
4a
116.1, qC
5
155.7, qC
6
108.0, CH
6.67, d (7.9)
4a, 5, 7, 8a
7
126.8, CH
7.09, d (7.9)
5, 8, 8a
8
119.7, qC
8a
157.9, qC
9
69.6, CH
4.70, m
7, 8
10
49.2, CH2
3.45, m
8, 9
7.30, s
11, 13, 14, 16
11
137.8, qC
12
133.1, CH
13
110.8, qC
14
157.1, qC
15
118.4, CH
6.77, d (7.2)
11, 13, 14, 16
16
128.8, CH
7.26, d (7.2)
11, 12, 14, 15
17
158.9, qC
18
101.4, CH
6.65, s
17, 19, 20, 22
19
156.9, qC
20
107.3, CH
6.74, d (7.8)
18, 19, 21, 22
21
130.5, CH
7.01, dd (7.6,7.8)
17, 19, 20, 22
22
106.8, CH
6.81, d (7.6)
17, 18, 20, 21
OH on C-5
9.26, br s
OH on C-14
8.48, br s
OH on C-19
9.47, br s
a
HMBC correlations, optimized for 8 Hz, are from proton(s) stated to the indicated carbon.
25
Table 3. NMR Spectroscopic Data of Ammonificin B (2) (400 MHz, DMSO-d6)
Position
C
H
HMBCa
2
70.2, CH2
4.45, m
8a, 4
3
83.3, CH
4.98, m
17
4
27.5, CH
4.35, d (5.6)
4a, 12, 16
4a
116.1, qC
5
155.7, qC
6
108.0, CH
6.67, d (7.9)
4a, 5, 7, 8a
7
126.8, CH
7.09, d (7.9)
5, 8, 8a
8
119.7, qC
8a
157.9, qC
9
69.6, CH
4.70, m
7, 8
10
49.2, CH2
3.45, m
8, 9
7.30, s
11, 13, 14, 16
11
144.2, qC
12
133.9, CH
13
126.9, qC
14
123.9, qC
15
132.5, CH
7.32, d (7.2)
11, 13, 14, 16
16
129.6, CH
7.28, d (7.2)
11, 12, 14, 15
17
158.9, qC
18
101.4, CH
6.65, s
17, 19, 20, 22
19
156.9, qC
20
107.3, CH
6.74, d (7.8)
18, 19, 21, 22
21
130.5, CH
7.01, dd (7.6,7.8)
17, 19, 20, 22
22
106.8, CH
6.81, d (7.6)
17, 18, 20, 21
OH on C-5
9.26, br s
OH on C-19
9.47, br s
a
HMBC correlations, optimized for 8 Hz, are from proton(s) stated to the indicated carbon.
26
Table of Contents Graphic
Ammonificins A and B, Hydroxyethylamine chroman derivatives from a Cultured Marine
Hydrothermal Vent Bacterium, Thermovibrio ammonificans.
Eric H. Andrianasolo, Liti Haramaty, Richard Rosario-Passapera, Kelly Bidle, Eileen White, Costantino Vetriani, Paul Falkowski
and Richard Lutz *
NH 2
OH
9
8
OH
8a
O
19
2
3
4a
5
HO
17
O
4
11
13
14
R
1 R= OH
2 R= Br
27
Br